Factor VIII (fVIII) participates in the intrinsic pathway of blood coagulation and is essential for normal hemostasis. The sites of hemostatically significant fVIII expression remain unknown and the control of fVIII expression is poorly understood. The problem is relevant to the basic biology of the hemostatic mechanism and to the development of better treatments for hemophilia A. Additionally, there is a clear association between elevated fVIII levels and thrombotic disease, which further underscores the need to understand the regulation of fVIII expression.This project has two specific aims. In Aim 1, we will study the endogenous synthesis of fVIII in vivo. We will identify cellular sites of murine fVIII synthesis by immunolocalization. Additionally, we will study the tissue distribution of fVIII using eGFP-fVIII transgenic mice. Human fulminant hepatic failure is associated with increased fVIII, which is surprising because the liver is considered the dominant site of fVIII synthesis. Understanding this phenomenon may be a key to identifying mechanisms of fVIII regulation. Therefore, we will study the tissue distribution of fVIII and fVIII mRNA in a murine model of fulminant hepatic failure. Transplantation studies have suggested that the spleen synthesizes hemostatically significant amounts of fVIII, but conflicting results have been obtained. Therefore, we will determine whether transplantation of normal donor spleen corrects the hemostatic defect in hemophilia A mice.In Aim 2, we will characterize the regulation of fVIII expression in heterologous systems. These systems are important models for the study of the regulation of fVIII synthesis. Additionally, heterologous expression is used in the commercial manufacture of fVIII. There is a worldwide shortage of fVIII that is due in part to low-level expression, in the last project period, we observed that expression of porcine B-domainless fVIII is approximately ten-fold higher than any level previously reported. We will identify sequences responsible for high-level expression of porcine fVIII. Additionally, we will identify the mechanism of differential expression of porcine versus human fVIII by using cell-free translation and co-translational processing systems.